CAN WE USE SYNTHETIC BIOLOGY AS AN

ALTERNATIVE TO PESTICIDES?

(An Unexpected Journey)

Keith G Davies

14 March 2018

Café Scientifique Hatfield

School to Rothamsted (10 years)from applied field ecology to the glasshouse

Rothamsted years (25 years)from the glasshouse to the laboratory

from the laboratory to the Eppendorf tube

from the Eppendorf tube to the genome

Structure of my talk

Post Rothamstedfrom the genome to synthetic biology

OPEC 1973 Oil Crisis

World Population< 4 billion (1975)

School to Rothamsted

One of 2 institutions at the time offering a degree with specialism in ecology

The only institution offering a degree in ecology with an industrial placement

School to Rothamsted

School to Rothamsted

European legislation regarding the use of pesticides

New legislation to replace Directive 91/414

A) New Registration regulation Directive 1107/2009B) Sustainable use Directive 2009/128/EC

Came into effect 14 June 2011

Also water framework directive

Priority to be given to non-chemical methods

Need for alternative nematode control methods

CONTEXT

School to Rothamsted

Rothamsted years

Plant-parasiticnematodes causelosses of around

$100 billionannually

C elegans movie

Bacterial wart diseasePasteuria penetrans

Increase in tomato yield following application of Pasteuriapenetrans to root-knot infected soils in Equador

adapted from Trudgill et al., (2000) Nematology 2, 823-845

Two Major Problems

1) Mass production

2) Infection and host specificity

Fallow Tomato Tomato

J2 per g Soil 94 64 7

%J2 +spores 47 89 100

Yield Kg/plot Na 11 25

Rhizoids

Granular masses

Penetration peg

Adhesion to cuticle

Sporulation

Matureendospores

Maturation

Rod

The life-cycle of Pasteuria penetrans: on root-knot Nematodes (Davies et al., Nematology 2011)

Meloidogyneincognita

Meloidogynearenaria

Reaction of second-stage juvenile cuticle by a polyclonalantibody

Davies & Danks Parasitology 105, 475-480

ENDOSPORE ATTACHMENT

Percentage of nematodes without Pasteuria spores in standard attachment bioassays with Meloidogyne arenaria ( ) and M. incognita ( )

Davies et al., 2001 Parasitology 122, 111-120

No fluorescence Low fluorescence High fluorescence

Davies et al., Let. Appl. Microb. 19, 370-373

Immunofluorescence of Pasteuria endospores

Immunofluorescence of Pasteuria endosporesattached to nematodes

Polyclonal antibody Monoclonal antibody

(Adapted from Preston et al., J. Nematology)

Cuticle characterisation using Mabs to Pasteuria

M. arenaria Race 1: peanutM. incognita Race 1: tobacco

Davies et al., Let. Appl. Microb. 19, 370-373

M. javanica: tobacco M. incognita Race 3: cotton

M. arenaria Race 1: peanutM. incognita Race 1: tobacco

M. incognita Race 3: cotton

Cuticle characterisation using Mabs to Pasteuria

Genomics

Caenorhabditis elegans Sequencing consortium 1998Meloidogyne incognita Abad et al., 2008Meloidogyne hapla Opperman et al 2008Heterodera glycines MonsantoBursaphenenchus xylophilus Kikuchi et al 2011Globodera pallida Urwin et al 2014Pratylenchus coffeae Opperman et al., 2015

Nematodes whole genomes

Endospore B. cereus(A. Moir)

Genomics

Caenorhabditis elegans Sequencing consortium 1998Meloidogyne incognita Abad et al., 2008Meloidogyne hapla Opperman et al 2008Heterodera glycines MonsantoBursaphenenchus xylophilus Kikuchi et al 2011Globodera pallida Urwin et al 2014Pratylenchus coffeae Opperman et al., 2015

Nematodes whole genomes

Selected Firmicutes whole genomes

Staphylococcus spp >100Clostridium spp >100Bacillus cereus >100Paenibacillus spp 51Bacillus subtilis 37Bacillus anthracis 38Bacillus thuringiensis 37

Endospore B. cereus(A. Moir)

Pasteuria genome survey sequences 4600

2 um

Spore attachment: Velcro-like mechanism

Davies, 2009, Advances in Parasitology 68, 211-245

A schematic diagram illustrating a possible model for the exosporium of the B. cereus family.

Kailas L et al. PNAS 2011;108:16014-16019

©2011 by National Academy of Sciences

0

0

41

41

B. anthracisStrain

770

818

Ames

53169

9602R, A2R, 6183R

6602

4229

5725R

G-x-y repeat region

Number FilamentG-x-y lengthrepeats (nm)

314 445 162 60.8

260 391 130 47.3

251 382 124 NA

239 370 118 48.7

131 262 52 28.4

122 253 48 29.8

113 244 42 25.1

92 223 28 13.9

GENOMICS & ENDOSPORE ATTACHMENT

Total of 12 Pasteuria penetrans collagens with G-X-Y repeats

5 unique to RES147, 4 unique toFl-1, 3 in common

79 G-X-Y repeats

62 G-X-Y repeats

36 G-X-Y repeats

GENOMICS & ENDOSPORE ATTACHMENT

>Contig1_2 (Ppenetrans-147)IFLGTTIGRLEFSGREFALGREGQQEHRGGPTTGPPGPPGAHGIQGPPGAQGIQGPAGTP

GAQGIQGPPGPAGTPGAQGIQGPPGPAGPAGPAGAAGSPGTPGPAGPAGPAGAAGSPGTP

GSPGTPGPAGPAGPAGPAGTPGTPGSPGTPGPAGPAGPAGTPGTPGAAGSPGTPGPAGPA

GPAGPAGTPGTPGPAGPQGTPGAPGPAGPQGTPGAPGPAGPQGTTGAAGPTGPQGTTGPQ

GTQGTQGPQGIQGIQGPVGPQGATGATGPGLNTSMTIVAGGGADTQFITPTPEGATGTAV

TLETGNGQRYGSGDIQLIGTTDILLPSTGTYLMSFHIDANYTSAAGAPVAGAYGSYVAYF

RQFTTDFFFNQIVAFWVGPALVNDAFDSSISNTVLGCVSDIPPHGLNNHMVRHESDVANS

LNTRRSQPQX

>Contig2_1 (Ppenetrans-147)

LQDQTNSPFRLEPEMWSINNIHSFNHMNKRKKQNIFFHTFSLGGLEDNHFMKHWIGRNSG

CIHYKNNGKIRNTITHTPSPRRSEGNRFVKHWIGRKSVYINSDYRDQHNHHNSSRTTLYR

NCEKCDNNQYEEFDNDHCEEFDNNHCCDCCLCNRCKCRVTGPTGPTGPTGRTGSTGRTGP

TGPTGRTGSTGRTGPTGPTGPTGPTGPTGRTGFTGRTGSTGSTGRTGPTGSTGRTGSTGS

TGRTGPTGSTGRTGSTGSTGRTGSTGSTSRPLVGRRNSR

>Contig3_4 (Ppenetrans-147)VANSRLEGWTAGPAGAQGISGPPGEPGIQGPAGTPGAQGIQGPPGPAGTPGAQGIQGPPG

PAGPTGPAGAAGSPGTPGPAGPAGPAGAAGSPGTPGSPGTPGPAGPAGPAGPAGTPGAPG

PAGPQGTPGAPGPAGPQGTPGAPGPAGPQGTTGAAGPTGPQGTTGPQGTQGTQGPQGIQG

IQGPVGPQGATGATGPGLNTSMTIVAGGGADTQFITPTPEGPTGPGGTFEPGNGPKYREG

GELHLIGTHRFSSSRVPGPF

The Future: Synthetic Biotechnolgy??

“… the real challenge will start when we enter the synthetic biology phase of research in our field. We will then devise new control elements and add these new modules to the existing genomes or build up wholly new genomes.”

Szybalski, 1974

Gibson et al., 2010 Science 329: 52-56

Within the synthetic genome Mycoplasma mycoidesthere were ‘watermarks’

CRAIGVENTER coded as:TTAACTAGCTAATGTCGTGCAATTGGAGTAGAGAACACAGAACGATTAACTAGCTAA

VENTERINSTITVTE coded as:TTAACTAGCTAAGTAGAAAACACCGAACGAATTAATTCTACGATTACCGTGACTGAGTTAACTAGCTAA

“WHAT I CANNOT BUILD, I CANNOT UNDERSTAND.”

– attributed to Richard Feynman

“TO LIVE, TO ERR, TO FALL, TO TRIUMPH, TO RECREATE LIFE OUT OF LIFE.”

– from James Joyce’s

The Future: Synthetic Biotechnolgy??

“… the real challenge will start when we enter the synthetic biology phase of research in our field. We will then devise new control elements and add these new modules to the existing genomes or build up wholly new genomes.”

Szybalski, 1974

Scie

nce

Mar

ch 2

5 2

01

6

Life-cycle disruption

(a) Hatch and migration• nematicides

• root diffusates• rhizosphere microorganisms

(b) Feeding, maturation and fecundity• nematicides

• host plant susceptibility• rhizosphere and endophytic micro-

organisms

(c) Mate finding and reproduction• nematicides

• mating disruption

Biological control perspective

(Gair et al., 1969)

Suppressive soilsare usually the

product of more than one organism

Davies et al., 1990

May June

July August

Nematodes Pasteuria Fungi

Cereal Cyst Nematode decline phenomenon

Fred Gommers

Biological control organisms

1) Fungal parasitesPochonia chlamydosporiaPaecilomyces lilacinusArthrobotrys oligosporaTrichoderma spp.

2) Bacterial parasitesPasteuria penetransRhizobacteriaBacillus spp.

Example 2 Arthrobotrys

Tunlid and Ahrén2011: In Keith Davies and Yitzak Spiegel (Eds) Biological Control of

Plant –Parasitic Nematodes

Peptides “nemin”elicitors

adhesins

Lectins

Serineproteases

Subtilisins

Toxic metabolites(linoleic acid)

Example 1 Pochonia

Kerry & Hirsch 2011: In Keith Davies and Yitzak Spiegel (Eds) Biological Control of

Plant –Parasitic Nematodes

Example 5 Pasteuria

Davies K G 2009, Adv. In Parasitol. 68, 211 - 245

Velcro – like attachment

Collagen moleculeslectins

Carbohydrates

Signalling pathways

Glycosyl-transferases

Mucins

Innate immunity

Designer biological control agents?

Lipopolysaccarhide effectors

Mucin effectors

Protease enzymes

Adhesion factors

Bacteriocins

Siderophores

Antifungal agents

Hormonal peptideschitinases

Signalling molecules

What is now proved was once only imaginedWilliam Blake

1757 - 1827

From Frankenstein toVenterstein?

“WHAT I CANNOT BUILD, I CANNOT UNDERSTAND.”

– attributed to Richard Feynman

“TO LIVE, TO ERR, TO FALL, TO TRIUMPH, TO RECREATE LIFE OUT OF LIFE.”

– from James Joyce’s

AcknowledgementsUNIVERSITY OF OXFORD

Jonathan HodgkinMaria Gravato-Nobre

Delia O’RoukeFreddie Partridge

Dave Stroud

BBSRC: Institute Development

Fellowship

Indian Agric. Res. Inst.Junaid Khan

Uma Rao

UC Davis: Valerie Williamson

George Brunning

NCSU: Charlie Opperman

David BirdBetsy Scholl

Indian Council Agric. Res. Arohi Srivastava

www.youtube.com/watch?v=sls1NzoaXZg